Liposomes are small vesicles composed of amphipathic lipids arranged in spherical bilayers. Liposomes are usually classified as small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), or multi-lamellar vesicles (MLV). SUVs and LUVs, by definition, have only one bilayer, whereas MLVs contain many concentric bilayers. Liposomes may be used to encapsulate various materials, by trapping hydrophilic compounds in the aqueous interior or between bilayers, or by trapping hydrophobic compounds within the bilayer.
Liposomes exhibit a wide variety of characteristics, depending upon their size, composition, and charge. For example, liposomes having a small percentage of unsaturated lipids tend to be slightly more permeable, while liposomes incorporating cholesterol or other sterols tend to be more rigid and less permeable. Liposomes may be positive, negative, or neutral in charge, depending on the hydrophilic group. For example, choline-based lipids impart a positive charge, phosphate and sulfate based lipids contribute a negative charge, and glycerol-based lipids and sterols are generally neutral in solution.
Liposomes have been employed to deliver biologically active material. See for example Allison, U.S. Pat. No. 4,053,585, which disclosed the administration of several antigens in negatively-charged liposomes, optionally including killed M. tuberculosis. Fullerton et al., U.S. Pat. No. 4,261,975, disclosed the use of separated influenza membranes, with hemagglutinin spikes attached, which is bound to liposomes for use in influenza vaccines.
Lipids having headgroups that are environmentally sensitive are desirable because the net charge of these molecules can be cationic, neutral, or anionic as dictated by the pH of the surrounding environment. Of particular interest are lipids with headgroups that are transiently cationic. Lipids with transiently cationic headgroups can convert into a non-lamellar phase upon a change in pH, and will deliver their contents into the cytoplasm. Cytoplasmic DNA delivery will enable high gene transfer. Transiently cationic lipids should also facilitate encapsulation of negatively charged nucleic acids, and promote the delivery of nucleic acids to the cytosol while maintaining low cytotoxicity and reduced immunoreactivity in vivo.
In addition to this transient cationic behavior, lipids that disperse in aqueous solution and form small (30-300 nm) bilayer structures are of interest as these lipids should be able to encapsulate small molecules as well as nucleic acids either by themselves or as a component in a liposomal formulation.
Lipid vesicles (liposomes) can be formed by a variety of techniques that, in general, start with “dry” lipids that are introduced into an aqueous phase (D. Lasic, J. Theor. Biol. (1987) 124:35-41). Once the lipid is hydrated, liposomes form spontaneously. Techniques have been developed to control the number of lamellae in the liposomes and to produce a defined particle size. The available procedures are satisfactory for most applications where small amounts of material are needed (G. Gregoriadis, “Liposome Technology” I-III (Boca Raton, Fla., CRC Press, Inc.), 1984). However, for the manufacture of vesicles on a large scale, the lipid hydration step can be a severe constraint on vesicle production. Furthermore, a method of synthesizing a liposome incorporating a zwitterionic lipid that allows for the reliable engineering of parameters such as liposome diameter, and amount of encapsulated bioactive substance encapsulated would represent an advance in the art. Accordingly, new methods for forming lipid vesicles are desirable.
Thus, there is a need in the art for lipids that are transiently cationic and methods of making encapsulents from these lipids. The present invention answers these and other needs.